Evaluating Mechanisms of Soil Microbiome Suppression of Striga Infection in Sorghum

The root parasitic weed Striga hermonthica has a devastating effect on sorghum and other cereal crops in Sub-Saharan Africa. Available Striga management strategies are rarely sufficient or not widely accessible or affordable. Identification of soil- or plant-associated microorganisms that interfere in the Striga infection cycle holds potential for development of complementary biological control measures. Such inoculants should be preferably based on microbes native to the regions of their application. We developed a method to assess microbiome-based soil suppressiveness to Striga with a minimal amount of field-collected soil. We previously used this method to identify the mechanisms of microbe-mediated suppression of Striga infection and to test individual microbial strains. Here, we present protocols to assess the functional potential of the soil microbiome and individual bacterial taxa that adversely affect Striga parasitism in sorghum via three major known suppression mechanisms. These methods can be further extended to other Striga hosts and other root parasitic weeds. Key features • This protocol provides a detailed description of the methods used in Kawa et al. [1]. • This protocol is optimized to assess soil suppressiveness to Striga infection by using natural field-collected soil and the same soil sterilized by gamma-radiation. • This protocol is optimized to test bacterial (and not fungal) isolates. • This protocol can be easily extended to other host–parasite–microbiome systems.

This protocol is used in: Cell Rep (2024), DOI: 10.1016/j.celrep.2024.113971 The root parasitic weed Striga hermonthica has a devastating effect on sorghum and other cereal crops in Sub-Saharan Africa.Available Striga management strategies are rarely sufficient or not widely accessible or affordable.Identification of soil-or plant-associated microorganisms that interfere in the Striga infection cycle holds potential for development of complementary biological control measures.Such inoculants should be preferably based on microbes native to the regions of their application.We developed a method to assess microbiome-based soil suppressiveness to Striga with a minimal amount of field-collected soil.We previously used this method to identify the mechanisms of microbe-mediated suppression of Striga infection and to test individual microbial strains.Here, we present protocols to assess the functional potential of the soil microbiome and individual bacterial taxa that adversely affect Striga parasitism in sorghum via three major known suppression mechanisms.These methods can be further extended to other Striga hosts and other root parasitic weeds.

Background
Sorghum (Sorghum bicolor) is among the five most important crops in the world and a staple food and forage cereal in Sub-Saharan Africa [2].Sorghum is grown in diverse agroecological zones, predominantly by small-hold farmers [3].Most sorghum cultivars are suited to arid, nutrient-depleted soils; yet, sorghum productivity is challenged by pathogens.The parasitic weed Striga hermonthica causes substantial sorghum yield losses, affecting approximately 60% of Sub-Saharan African farmlands [4].Striga is a root parasite that infects cereal crop species, including sorghum, rice, and pearl millet [5].Striga's life cycle is dependent on specific compounds exuded from the host roots into the surrounding soil.Striga germinates upon the perception of strigolactones, while haustorium-inducing factors (HIFs) initiate the development of a haustorium, a specialized organ that allows Striga to attach to and penetrate the root tissue [6][7][8].Upon reaching the host vasculature, Striga connects its own xylem vessels with its host vasculature [7], enabling the parasite to withdraw water and nutrients from its host, thereby compromising host fitness and productivity [9].Striga management includes manual weed removal, chemical control methods, and breeding for host resistance [10].Chemical application poses a challenge in rain-fed agricultural systems, while commercial sorghum varieties show only partial resistance and are not always accessible or suited to the agricultural practices of small-holder farmers [11].Recently, the functional potential of the soil microbiome to suppress Striga infection has been described.These include microbial isolates that are pathogenic to Striga [12] and those that degrade HIFs or that induce physical barriers to Striga parasitism in host roots [1].Further development of microbial-based agricultural solutions against Striga will require screening candidate microbes native to soils in Striga-infested regions.
Here, we present a set of protocols to assess the contribution of soil microbiome to Striga infection levels and individual soil-borne bacterial isolates to Striga suppression-associated mechanisms.The soil plug assay enables testing Striga suppressiveness of multiple soils collected from agricultural fields.Striga resistance modes are typically described as pre-attachment resistance [reduced germination and (pre)haustorium formation], and postattachment resistance (when parasite fails to penetrate the root tissue and/or establish the vascular connection with the host).To distinguish which stage of Striga infection the soil microbiome affects, we recommend extracting and testing host root exudates in an in vitro Striga germination assay and a haustorium formation assay.Preparation of host root cross-sections and their histological staining allows quantification of aerenchyma and suberization, both associated with microbe-mediated suppression of post-attachment stages of Striga infection [1].To test which isolates affect haustorium formation and induce changes in host root cellular anatomy, we present an in vitro haustorium induction assay and a method of sorghum inoculation in sand, respectively.The presented protocols can be easily adapted and applied to multiple sorghum cultivars and other Striga hosts.e. Seal with parafilm and return to 30 °C in a dark incubator.f.After three days, count the total number of seeds and number of germinated seeds in the viewing area using a stereomicroscope.g.Calculate the germination rate for each Petri dish.GR% = (Ngs/Nts) × 100 Ngs: Total number of germinated seeds per Petri dish Nts: Total number of seeds per Petri dish 7. Use these estimates to treat each experiment with the desired amount of germinable Striga seeds using the following formula:

C. Soil plug assay for soil suppressiveness
C1. Soil sterilization 1. Air-dry the soil at room temperature for 4-7 days.2. Sieve the soil through a 4 mm mesh.3. Sterilize the soil by gamma irradiation with an 8 kGy dose.Notes: a.To attribute the Striga suppressive effect solely to its microbiome, it should be ensured that the physicochemical properties of gamma-sterilized and natural (non-sterilized) soil remain comparable.b.Assess bacterial and fungal diversity to ensure which is depleted.

C2. Soil plug preparation
1. Mix each soil batch (sterilized and non-sterilized) with sterile water to reach 5% of moisture level (w/v).2. Cut a hole at the bottom of a 50 mL conical tube to provide drainage of excess liquid.3. Wrap the tubes in aluminum foil or other comparable material to block light.Do not cover the hole at the bottom.4. Fill the tubes with soil. 5.With a 1 mL pipette tip, make a hole in the soil for a germinated sorghum seedling.6.Using forceps, gently place a 3-day-old sorghum seedling in the soil.7. Place sorghum in the greenhouse or grow in a chamber at 28 C during the day (11 h) and 25 C at night (13 h), with a light intensity of 450 mol/m 2 /s and 70% relative humidity.8. Apply 3 mL of sterile deionized water to each soil plug every second day.9. Grow sorghum in the soil plug for 10 days.

C3. Cone preparation
1. Sterilize 3,000 germinable Striga seeds per plant as determined in step B5. 2. Autoclave the cones prior to planting.3. Place a gauze pad to cover the hole at the bottom of each cone and secure it with a rubber band.4. Fill each cone with 350 mL of fresh (not preconditioned) sand (Figure 1A). 5. Top up with 350 mL of preconditioned sand without (control) or with Striga seeds.6.Within the 50 mL conical cone, make a hole to accommodate the soil plug.7. Transfer the 10-day-old sorghum seedling together with the soil plug to the cone (Figure 1A).8. Cover the soil plug surface with a thin layer of fresh sand.9. Place sorghum in the greenhouse or growth chamber at 28 C during the day (11 h) and 25 C at night ( 13h) with a light intensity of 450 mol/m 2 /s and 70% relative humidity.10.Apply 50 mL of sterile half-strength modified Hoagland solution to each plant on days 0, 7, and 14.Apply 50 mL of sterile deionized water to each plant on days 1, 4, 10, 13, and 17. 11.Quantify Striga infection 14 and/or 21 days after transfer to cones.2. Shake the plant to remove as much of the sand and soil from the roots as possible.Collect all the soil and sand.3. Gently swirl the roots in a tray filled with water to wash off the remaining substrate.Do not rub the roots, since Striga might get de-attached from the host.4. Gently dry the roots with a paper towel.5. Cut the root system from the shoot.6. Record the root's fresh weight.7. Place the root system in a transparent tray filled with water.8. Use a stereomicroscope with 10× magnification to examine the roots for sites of Striga attachment and penetration.Use forceps to spread the roots and systematically look for the presence of Striga.Refer to Figures 2D and 2E as to how to distinguish attached from penetrated Striga.9. Screen the sand and soil collected in step D2 for any attached Striga that may have fallen off the root, called "de-attached" Striga (see Figure 2C for the developed, penetrated Striga that de-attached from sorghum root).10.Sum the number of penetrated Striga on the roots and the number of Striga rescued from the sand ("deattached Striga") to obtain the total penetration number.11.Sum the number of total penetrated Striga and attached Striga to obtain the total Striga count.12.The total Striga count and the total number of attachments should be normalized by the fresh weight of the root scored in step D6.

Figure 1 .
Figure 1.Growth setup to assess soil suppressiveness to Striga in soil plug cone assay (A) and screening for bacterial isolates associated with Striga-suppressive phenotypes (B)

Figure 2 . 1 .
Figure 2. Striga developmental stages of interest.(A) Striga germination and (B) haustorium development of in vitro experiments.(C) Striga may break off at the haustorial connection during harvest even when well-developed.(D) Both attachment and penetrated Striga have developed haustoria that are connected to the sorghum root, but the penetrated Striga shows a further development of leaf lobes.Yellow arrowheads: developing leaf lobes in young, penetrated Striga.(E) Older penetrated Striga may begin developing green leaf tissue.

Biological materials Recipes 1. Half-strength modified Hoagland media
Published:

Procedure A. Sorghum seed surface sterilization and germination
Note: Always try to use sorghum seeds that are similar in size.1.Place seeds in a 50 mL conical tube.2.Add up to 50 mL of freshly prepared sterilizing solution (30% commercial bleach, 0.2% Tween-20, v/v).3.Gently agitate seeds on an orbital shaker in sterilizing solution for 20 min.4. Discard the sterilizing solution and seeds that float on the surface of the solution.5. Wash seeds with sterile water for 2 min, five times.6. Optional: If fungal contamination occurs, agitate seeds overnight in 5% (w/v) Captan slurry followed by five washes in sterile water.7. Place seeds on sterile Petri dishes containing two Whatman filter papers moistened with 5 mL of sterile water.8. Seal plates with parafilm.9. Incubate plates in the dark at 30 °C for the duration indicated per experiment. B.

Striga seed sterilization and preconditioning 1
. Estimate the density of Striga seeds.a.Weigh out a small number of dry seeds on a Whatman filter paper in a Petri dish.b.Record mass.c.Count the total number of seeds using a stereomicroscope.d.Calculate the number of seeds per milligram.e.Repeat steps B1a-d at least three times and calculate an average.Note: We recommend conducting this once for each new batch of Striga seeds.2.Wash Striga seeds in 10% (v/v) bleach and 0.02% (v/v) Tween-20 for 10 min.3.Discard the solution and wash the seeds five times in sterile water.4.Precondition Striga seeds by incubation at 30 C in the dark.The chemicals and protocols needed for preconditioning are dependent on the experiment and are outlined in their respective sections.5.Optional: The number of Striga seeds for each experiment is indicated as the number of germinable seeds.For each newly collected Striga seed batch, germinability should be assessed, as it is highly variable.6. Calculate Striga germinability: a. Sterilize a specific amount (number or weight) of Striga seeds as determined in step B1. b.Disperse seeds onto a Petri dish with two sterilized Whatman filter papers moistened with 5 mL of sterile water.c.Precondition at 30 C in the dark for 10-14 days.d.Transfer seeds to a new Petri dish with two sterile Whatman filter papers moistened with 5 mL of 1 ppm GR24rac.
Cite as: Taylor, T. et al. (2024).Evaluating Mechanisms of Soil Microbiome Suppression of Striga Infection in Sorghum.Bio-protocol 14(17): e5058.DOI: 10.21769/BioProtoc.5058.6 Published: Sep 05, 2024 seeds required (mg) = number of germinable seeds required per plant Striga seeds germination percentage × number Striga seeds in 1mg × number of sorghum plants to be infected a. Calculate the total amount of sand needed (you can use 1/10 of required sand amount for preconditioning and mix with the remaining 9/10 on the day of planting).b.Mix sand with sterile water to reach approximately 16% (v/w) moisture level.c.Add Striga seeds to wet sand and mix thoroughly.d.Incubate sand-Striga mix at 30 C in the dark for 10-14 days.e. Prepare the sand used as a negative control in the same manner, without adding the Striga seeds.